PROJECT SUMMARY Gastrointestinal diseases caused by Salmonella enterica serovar Tymphimurium (S. Typhimurium) lead to numerous illnesses, hospitalizations, and deaths globally every year. S. Typhimurium successfully invades host cells by injecting virulence effectors that modulate the host actin cytoskeletal network, through host cell machinery such as Rho GTPases. The Rho GTPase family member Cdc42 has been identified as a target of S. Typhimurium; however, specific mechanisms for its recruitment and its function in vivo during a Salmonella infection remain unclear. Preliminary studies demonstrate that Cdc42 Effector Protein 1 (Cdc42EP1), a novel binding protein isolated from Cdc42 interactome, localizes to Salmonella entry sites in epithelial cells. Additionally, its interaction with Cdc42 variants deficient of their lipid modifications is reduced. Although S. Typhimurium has been found to utilize host lipidation enzymes to modify its own proteins, it is unknown how the prenylation and palmitoylation modifications of Cdc42 impact Salmonella?s invasive ability. We hypothesize that S. Typhimurium recruits Cdc42 via Cdc42EP1, and that altering Cdc42?s lipid modifications impacts bacterial invasion and disease susceptibility in vivo. Two aims are proposed to determine the cellular function and impact of Cdc42EP1-Cdc42 machinery during Salmonella infection in vitro and in vivo. The specific goal of this project is to understand whether Salmonella exploits Cdc42EP1-Cdc42 machinery to gain entry and survival in host cells, and how this cellular machinery impacts the pathogen?s invasive ability in vivo. Aim 1 will address the molecular mechanisms by which S. Typhimurium manipulate and recruit both Cdc42 and Cdc42EP1 during invasion. The role of Cdc42EP1 during Salmonella invasion, its spatial and temporal relationship with Cdc42 upon infection, and its interaction with lipidation-deficient Cdc42 mutants during infection will all be studied. Aim 2 will utilize our unique genetically engineered Cdc42 mouse models to determine whether altering Cdc42 machinery affects the susceptibility to a Salmonella infection in vivo. Cdc42 knockout mice will be used to determine how deletion of Cdc42 impacts Salmonella invasion. We will also utilize our newly developed transgenic mice that express palmitoylable Cdc42 to understand how this modification may impact a bacterial infection and disease susceptibility. In addition to analysis of bacterial invasion in these mice, we will determine if Salmonella modify the host protein using an acyl biotin exchange assay. Together, these aims will reveal the mechanism of Salmonella-Cdc42 interaction at cell biology and physiology levels.